Prolactin is a peptide hormone of 199 amino acids which is produced by the anterior pituitary gland. This hormone has a variety of biological activities in mammals, including lactogenesis and modulation of the immune system. Receptors for prolactin have been found on over fifty different cell types. A number of variants of the hormone have been identified. These include post-translational modifications such as glycosylation, phosphorylation, disulfide dimerization and proteolytic cleavages, as well as alternatively spliced forms. The biological role of these variants is not fully understood. It is postulated that different variants of the molecule may be responsible for the diverse physiological actions of prolactin. In humans, the primary form in the pituitary gland is non-glycosylated, while the glycosylated form predominates in circulation.
The method most commonly used to separate glycosylated prolactin from non-glycosylated prolactin is lectin affinity chromatography. This technique is used in the purification of prolactin from many sources. These include: glycosylated human prolactin from pituitary explants (Lewis et al. Endocrinology, 124: 1558-1563, 1989), glycosylated ovine prolactin from pituitary explants (Lewis et al., Proc. Natl. Acad. Sci., 81: 203-215, 1991), glycosylated porcine prolactin from pituitary explants (Sinha et al., Mol. Cell. Endocrinology, 80: 203-215, 1988) and human glycosylated prolactin from cultured prolactinoma cells (Pellegrini et al., Endocrinology, 122: 2667-2674, 1988). The most commonly used lectins are Concanavalin A (Con A) and lens culinaris (lentil).
The use of reverse phase chromatography in the separation of glycosylated and non-glycosylated prolactin has also been reported. See Noso et al., Int. J. Peptide Protein Res., 39: 250-257, 1992.
The lectin chromatography method of prolactin purification presents binding specificity problems. Both naturally occurring and recombinant glycoproteins will often be produced with a heterogenous range of attached oligosaccharide chains. Due to their binding specificity, a given lectin may not bind all of the oligosaccharide forms present. In fact, a number of investigators have reported working with forms of naturally-derived glycosylated prolactin which will not bind to the particular lectin being used for purification.
The low efficiency of lectin affinity chromatography makes it a poor choice for preparative scale processes. Lectin affinity resins typically have a low binding capacity. This makes purification processes cumbersome and may also lead to the contamination of non-glycosylated fractions with glycosylated prolactin. In addition, lectin resins are not typically available on rigid matrixes which will support the high flow rates which are desirable for production scale purification. A further complicating factor is that the lectin proteins can leach off the column during purification. Further purification steps may be needed since lectins are often highly toxic and would complicate in vivo studies.
Reverse phase chromatography also presents drawbacks in the separation of glycosylated and non-glycosylated proteins since it relies upon the use of high concentrations of acetonitrile and may also utilize strong acids such as trifluoroacetic acid (TFA). The use of these solvents and acids may have an adverse effect upon protein function.